The key signal that contracts bladder smooth muscle is an increase in [Ca]i which activates Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), thereby promoting phosphorylation of myosin regulatory light chain (RLC) and initiating contraction. Emerging evidence suggests that functional derangements associated with chronic obstruction of the urinary bladder are associated with significant changes in the contractile protein signaling system. We propose to investigate mechanisms necessary for activation of the bladder smooth muscle contractile apparatus to unravel the complexities of interacting signaling networks. Specific Aim 1. Is MLCK the only kinase that phosphorylates RLC in a Ca -dependent manner in bladder smooth muscle? Transgenic mice expressing biosensor MLCK will be used to determine Ca2+-dependency of activation relative to [Ca2+]i, RLC phosphorylation and contraction. Protein transduction domain inhibitors will be used to test involvement of specific kinases. Smooth muscle MLCK gene ablation will be restricted to smooth muscle cells by a tamoxifen-controlled ablation system. Contractile responsiveness will be measured in isolated bladder tissues and in vivo. Specific Aim 2. Is MLCK targeting to the contractile domain of smooth muscle cells necessary and sufficient for RLC phosphorylation? Gene exons containing the actin-binding motifs and the myosin binding module will be deleted by knock-in procedures and contractile performance of bladder smooth muscle will be characterized in vitro and in vivo. Specific Aim 3. What are the roles ofnonmuscle myosin in smooth muscle? Is it regulated selectively by Rho kinase and PKC? Blebbistatin, a selective inhibitor of nonmuscle myosin II will be used to evaluate contributions of nonmuscle myosin to contractile properties. Nonmuscle RLC phosphorylation will be measured along with smooth muscle RLC under different modes of signaling. We consider the possibility that nonmuscle myosin may contribute to the stability of membrane adhesion sites necessary for cellular force transmission by intracellular contractile domains in bladder myocytes.